1. Field of the Invention
The present invention relates to methods for preventing and/or treating diseases caused by an inflammatory response mediated by endogenous substance P. The methods comprise the administration to a subject of a pharmaceutically-effective amount of anti-substance P antibodies, or anti-substance P antibody fragments, thereby inhibiting the activity of endogenous substance P. Such inhibition reduces the levels of cytokines produced by T lymphocytes, alters the signals which direct the inflammatory response to the infection, and reduces cytokine-induced inflammation. The present invention especially relates to methods for preventing and treating diseases caused by an inflammatory response to viral or bacterial infections. Moreover, the present invention especially relates to methods for preventing and treating diseases caused by an inflammatory response to respiratory syncytial virus.
2. Background
Inflammation. Inflammation is a localized, protective response of the immune system which is produced to protect a subject from disease. It is elicited by destruction of tissues, foreign substances such as allergens, infectious agents and cells such as viruses and bacteria. The inflammatory response destroys, dilutes or sequesters both the injurious agent and the injured tissue. It is characterized in the acute form by the classical signs of pain, heat, redness, swelling and loss of function. Histologically, it involves a complex series of events, including dilatation of arterioles, capillaries and venules, with increased permeability and blood flow, exudation of fluids, including plasma proteins, and leukocytic migration into the inflammatory focus.
T lymphocytes, which are products of lymphoid tissue, and their soluble protein products, known as cytokines, participate in humoral and cell-mediated immunity, and mediate the inflammatory response. T lymphocytes can suppress or assist the stimulation of antibody production in the presence of antigen, and can kill tumor and transplant tissue cells. Cytokines made by T lymphocytes, such as the intracellular cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-γ (IFNγ), function to augment or suppress the immune response, or to change the type of immune response.
The immune-system-mediated inflammatory response, however, can also cause, or contribute to, disease, rather than prevent it. Some diseases, such as those caused by infection by respiratory syncytial virus appear to be caused in part by the inflammatory response. Small molecule anti-inflammatory agents which are currently employed to treat inflammation, disadvantageously have adverse side effects, such as gastrointestinal discomfort and decreased blood clotting efficiency. Steroid-based anti-inflammatory drugs also have adverse side effects, such as reduced adrenal gland function and generalized immune system inhibition.
Substance P. Substance P is a naturally-occurring, endogenously-released, undecapeptide which has multiple sites of action as a major pro-inflammatory neuromediator or neuromodulator, both in the central and peripheral nervous systems. Substance P is known to have regulatory effects upon several cell types present in the immune system, and to regulate the inflammatory response to foreign substances or pathogens by altering the expression of proinflammatory cytokines, which have been implicated in the pathogenesis of different neuropathologies. High levels of substance P have been implicated in the pathogenesis of diseases associated with inflammation. Substance P is also known to modulate the activity of cells affiliated with respiratory inflammation (neutrophils, mast cells and alveolar macrophages). Elevated concentrations of substance P and cytokines have been associated in the pathogenesis of a wide variety of diseases, including arthritis, experimental allergic encephalomyelitis, altered immunoregulation during human immunodeficiency virus infection, irritable bowel syndrome and airway hyperactivity.
Substance P has the following amino acid sequence (SEQ ID NO:1 in the Sequence Listing):
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2,
and belongs to a family of closely-related peptides known as the tachykinins (neurokinins), along with two other related, endogenously-released peptides, neurokinin A (NKA) and neurokinin B (NKB). Neurokinins are a group of structurally-related peptides characterized by a similar C-terminal pentapeptide that interacts with different receptor subtypes. The amidated C-terminal portion of the substance P neuropeptide is responsible for receptor binding (Otsuka et al., “Neurotransmitter Functions of Mammalian-Tachykinins,” Physiological Reviews 73:229–307 (1993)). Substance P and neurokinin A are widely distributed in the airways and lungs of several species, including humans and guinea pigs.
Substance P is known to participate in inflammatory processes, and in the immune response, and appears to be involved in the process of neurogenic inflammation, pain transmission, regulation of blood pressure, inflammation processes and endocrine and exocrine secretion. Substance P has been strongly implicated in the transmission of pain, being coreleased alongside other transmitters and peptides, from small-diameter primary afferents. The primary role of substance P in neurogenic inflammation, smooth muscle contraction, and vasodilation is well established. In the central nervous system, where substance P and its receptors are widely distributed, its functional roles are less clear. Given parenterally, substance P and neurokinin A induce a variety of responses, including contraction of bronchial smooth muscle, mucus secretion, vasodilation, extravasation of plasma proteins and recruitment of inflammatory cells. The principal biological activity of these tachykinins resides in their structurally similar carboxyl sequence, . . . Phe-X-Gly-Leu-Met-COOH (SEQ ID NO. 2). Several binding experiments have shown that intestinal and vascular substance P receptors bind to the C-terminal pentapeptide of substance P. It has been suggested that the release of endogenous substance P and neurokinin A from pulmonary afferent C-fibers contributes to bronchial obstruction in asthma.
Specific membrane receptors for substance P have been found in neurons, and in muscular, glandular and immunocompetent cells, which helps to explain the diversity and importance of the physiological roles of substance P. Substance P effects its biological activity by ligation with these receptors, and the binding of substance P to its receptors enhances the incorporation of phosphate or inositol into phosphatidylinositol. Substance P, neurokinin A and neurokinin B appear to exert their biological effects primarily through interactions with three distinct receptors of the neurokinin family, named neurokinin receptor-1 (NK1), neurokinin receptor-2 (NK2) and neurokinin receptor-3 (NK3), respectively. Substance P affects antibody synthesis, or promotes cytokine production, through NK1 receptors which are well-characterized on T- and B-lymphocytes, monocytes and astrocytes, and possibly also via nonneurokinin receptors. While substance P preferentially binds to the NK1 receptor, its binding is not limited to this receptor. Further, the cationic, amphiphilic nature of the substance P molecule allows it to bypass the NK1 receptor and insert itself into the cell membrane, where it can directly interact with proteins. Thus, any inflammation-reduction therapy which targets the NK1 receptors (i.e., the use of compound or antibody NK1 receptor antagonists) will not fully inhibit the substance P pathway of inflammation, or be effective in preventing the pro-inflammatory actions of substance P.
The functional roles of substance P in the mammalian central nervous system and peripheral nervous system are currently being investigated by a variety of experimental approaches, including the use of antibodies and pharmacological agonists and antagonists. However, a continuous delivery of pharmaceutical drugs to the nervous tissue poses several practical problems, including limited diffusion and half-life of the drugs, secondary, non-specific effects of the drugs (because the endogenous neurokinins are preferential, but not selective agonists) and side-effects of the drugs. While studies have been performed with pharmaceutical antagonists of substance P receptors, studies in this area have been limited by the lack of efficacious antagonists specific for the NK1 receptors, and by the difficulty of using these antagonists in vivo, due to short half-lives, and the resulting lack of effect, and non-specific effects. Thus, the use of antibodies and antibody fragments which bind directly with substance P, as is done in the methods of the present invention, rather than using substance P receptor antagonists, is believed to be a preferable approach for inhibiting the action of endogenous substance P and, consequently for reducing the levels of cytokine production and cytokine-induced inflammation.
Additional information concerning substance P is present in the following publications: U.S. Pat. No. 4,680,283; Mantyh, “Substance P and the Inflammatory and Immune Response,” Annals of the New York Academy of Sciences 632:263–271 (1991); Ho et al., “Substance P Modulates Human Immunodeficiency Virus Replication in Human Peripheral Blood Monocyte-Derived Macrophages,” AIDS Research & Human Retroviruses 12:195 (1996); Kudlacz et al., “Parainfluenza Virus Type 3 Induced Alterations in Tachykinin NK1 Receptors, Substance P Levels and Respiratory Functions in Guinea Pig Airways,” European Journal of Pharmacology 270:291 (1994); Yamawaki et al., “Viral Infection Potentiates the Increase in Airway Blood Flow Produced by Substance P,” Journal of Applied Physiology 79:398 (1995); Ben-Jebria et al., “Effect of Passive Sensitization on the Mechanical Activity of Human Isolated Bronchial Smooth Muscle Induced by Substance P, Neurokinin A and VIP,” British Journal of Pharmacology 109:131 (1993); Boichot et al., “Inhaled substance P Induces Activation of Alveolar Macrophages and Increases Airway Responses in the Guinea-Pig,” Neuropeptides 25:307 (1993); Cheung et al., “Effects of Inhaled Substance P on Airway Responsiveness to Methacholine in Asthmatic Subjects In Vivo,” Journal of Applied Physiology 77:1325 (1994); Heaney et al., “Substance P induces Histamine Release from Human Pulmonary Mast Cells,” Clinical & Experimental Allergy 25:179 (1995); Murris-Espin et al., “Substance P and Alveolar Macrophages: Effects on Oxidative Metabolism and Eicosanoid Production.” Allergy 50:334 (1995); Tomaki et al., “Elevated Substance P Content in Induced Sputum from Patients with Asthma and Patients with Chronic Bronchitis,” American Journal of Respiratory & Critical Care Medicine 151:613 (1995); Yoshihara et al., “Involvement of Substance P in the Paroxysmal Cough of Pertussis,” Regulatory Peptides 46:238 (1993); Ahmed et al., “Capsaicin Effects on Substance P and CGRP in Rat Adjuvant Arthritis,” Regulatory Peptides 55:85 (1995); Jarrah et al., “Cholecystokinin-Octapeptide (CCK-OP) and Substance P(SP) Influence Immune Response to Cholera Toxin in Live Animals,” Advances in Experimental Medicine & Biology 371A:563 (1995); Kincy-Cain et al., “Substance P-Induced IL-12 Production by Murine Macrophages,” Journal of Immunology 158:2334 (1997); Palma et al., “Interleukin-6 Production by U373 MG, a Human Astrocytoma Cell Line; Different Pathways Involved in Substance P and Lipopolysaccharide Activation,” Journal of Immunology 59:155 (1995); and Shadiack et al., “Lipopolysaccharide Induces Substance P in Sympathetic Ganglia Via Ganglionic Interleukin-1 Production,” Journal of Neuroimmunology 49:51 (1994).
Respiratory Syncytial Virus. Respiratory syncytial virus is one example of an agent which causes an infection which, in turn, often results in a disease produced by an inflammatory response to the infection that may be mediated by endogenous substance P. The immune response to respiratory syncytial virus infection is characterized by the enhanced production of cytokines, increased levels of substance P, and bronchial inflammation. Respiratory syncytial virus is the most important cause of pneumonia and bronchiolitis in infants, and can result in death.
The only available treatment for respiratory syncytial virus is ribavirin, a purine nucleoside analog which inhibits the replication of a wide range of RNA and DNA viruses, and which has many disadvantages. The administration of ribavirin to patients in the form of an aerosol is costly and difficult, and limits its therapeutic efficacy. Furthermore, ribavirin antagonizes the activity of the antiviral agent zidovudine against human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS. When administered orally or intravenously, ribavirin causes anemia due to extravascular hemolysis and suppression of the bone marrow. Further, ribavirin is mutagenic in small animals, and the oral bioavailability of ribavirin is only about 45 percent. The long-term oral therapy of ribavirin is associated with both gastrointestinal and adverse symptoms in the central nervous system.
A need presently exists for an efficacious and convenient method for treating and preventing diseases which are caused by an inflammatory response mediated by substance P. Accordingly, the present invention provides an effective and convenient method for preventing and treating diseases caused by an inflammatory response to a variety of conditions that is mediated by substance P comprising inhibiting the activity of endogenous substance P with anti-substance P antibodies and/or anti-substance P antibody fragments. The data presented in the examples set forth below show that the inhibition of the biological activity of substance P with anti-substance P F(ab)2 antibody fragments reduces the production of cytokines by T lymphocytes, thereby altering the signals which direct the inflammatory response to an infection caused by respiratory syncytial virus, and reducing potential cytokine-induced inflammation.
In accordance with the methods of the present invention, blocking the activity of endogenous substance P can potentially be employed to prevent or treat a wide variety of diseases or syndromes caused in whole or part by an inflammatory response mediated by substance P. Such diseases or syndromes include, by way of nonlimiting example, diseases ascribable to viral or bacterial infection such as infections associated with inflammatory bowel disease, virus-mediated bronchiolitis including that mediated by respiratory syncytial virus, bacterial colitis, inflammation associated with chlamydial diseases, lung injury associated with staphylococcal enterotoxin B, inflammation due to cytomegalovirus or hepatitis B virus, and sepsis, allergic diseases such as asthma, autoimmune diseases such as rheumatoid arthritis, pancreatitis, and inflammation associated with multiple sclerosis, and rejection of allografts and other transplanted tissues or organs. The prevention, reduction or elimination of the substance P-mediated inflammatory response can also be used to prevent adverse effects which may otherwise result from tissue or organ transplantation.
The administration of anti-substance P antibodies, or anti-substance P antibody fragments, to a subject to inhibit the activity of endogenous substance P is preferential to the administration of substance P NK1 receptor antagonists to the subject because, unlike substance P NK1 receptor antagonists, anti-substance P antibodies and anti-substance P antibody fragments inhibit the activity of substance P without receptor ligation, and before the substance P can cross the cell membrane in a receptor-independent manner.